The role of the interaction between the C-terminal domain of RNA pol II and a histone methyltransferase in mediating epigenetic memory in malaria virulence genes Plasmodium falciparum is responsible for the most acute and severe form of human malaria. This protozoan parasite infects the circulating red blood cells of its human host, placing on the infected cell surface the primary virulence and antigenic determinant, an adhesive protein called PfEMP1. Different forms of PfEMP1 are encoded by individual members of the large, multicopy var gene family. Only a single var gene is expressed at a time. Switching which var gene is expressed aids the parasite in its ability to evade clearance by the immune system and is referred to as antigenic variation. The mechanism by which the expression of individual var genes is regulated is poorly understood, however recent work has demonstrated that histone modifications play a significant role. Once a var gene is activated, it tends to remain active for many cellular divisions, a property referred to as "epigenetic memory." Studies have also shown that active transcription by RNA polymerase II is required for the maintenance of epigenetic memory of P. falciparum var genes, suggesting that the polymerase itself might play a role in maintaining the necessary chromatin modifications required for regulating var gene expression. In addition, it was recently shown that rodent parasites do not utilize epigenetic memory to control the expression of their virulence genes. Phylogenetic comparisons reveal an unusual expansion of the C-terminal domain (CTD) of the largest subunit of RNA pol II of primate parasites that is not found in parasites of rodents, birds or bats. Further, this region of RNA pol II has been shown to interact with chromatin modifying enzymes in higher eukaryotes, suggesting that it could be involved in maintenance of epigenetic memory. Interestingly, two specific histone modifying proteins, the histone methyltransferase PfSet2 and its cognate demethylase PfJmjC1, are similarly only found in primate parasites. Previous structural and biochemical work in yeast has shown that the phosphorylated form of the RNA pol II CTD can directly interact with Set2 to recruit it to actively transcribed genes, thus helping to maintain active chromatin at highly transcribed regions of the genome. Our hypothesis is that PfSet2 is recruited to transcriptionally active var genes through interactions with the phosphorylated CTD of P. falciparum RNA pol II to enforce epigenetic memory. The goal of this study is to determine if PfSet2 directly interacts with the CTD using biochemical and structural approaches, and to investigate the role of this interaction in the maintenance of epigenetic memory of var genes by introducing mutant forms of PfSet2 in P. falciparum. The long term goal is to determine the role of the interaction between the CTD and PfSet2 in promoting virulent mechanisms such as antigenic variation in P. falciparum. PUBLIC HEALTH RELEVANCE: Each year, millions of people worldwide are afflicted with P. falciparum malaria resulting in approximately a million deaths of mostly young children in sub-Saharan Africa. Previous work demonstrated that placement of an antigenic determinant called PfEMP1 on human red blood cells is essential for P. falciparum survival, and is regulated by a var gene switching mechanism that aids the parasite in its ability to evade the immune system. Understanding the regulation of this gene switching mechanism can lead to the development of more efficient drugs and possibly a vaccine against this acute and severe disease.